The toxicity of NPs is not well characterized in terms of their size. In particular, the size-based toxicity of fullerene (C60) remains an issue because of a lack of C60 NPs with a well-controlled size. In this work, six fractions of the nano-C60 aggregates (nC60) with different size distribution were prepared by a simple differential centrifugation. By using these nC60fractions, we demonstrate the size-dependent inhibition of DNA polymerase and reduced-size enhanced cytotoxicity. Above all, we found that nC60 NPs with smaller size may have higher toxicity potency. These size-dependent effects were observed at the high exposure doses (4–6 mg/L). Interestingly, at 20-times lower and noncytotoxic doses, the size-dependent effect can be indicated by apoptosis-related fluorescent protein fused Bax translocation. Considering the toxicity of NPs is often ignored in the traditional end-point analysis for cytotoxicity when the exposure dose is low, the findings presented here will assist in the evaluation of the size-dependent cytotoxicity and dose–response relationships of toxicity mediated by nC60 NPs at low doses.